The ATR Inhibitor VE-821 Enhances the Radiosensitivity and Suppresses DNA Repair Mechanisms of Human Chondrosarcoma Cells.

To overcome the resistance to radiotherapy in chondrosarcomas, the prevention of efficient DNA repair with an additional treatment was explored for particle beams as well as reference X-ray irradiation. The combined treatment with DNA repair inhibitors-with a focus on ATRi VE-821-and proton or carbon ions irradiation was investigated regarding cell viability, proliferation, cell cycle distribution, MAPK phosphorylation, and the expression of key DNA repair genes in two human chondrosarcoma cell lines. Pre-treatment with the PARPis Olaparib or Veliparib, the ATMi Ku-55933, and the ATRi VE-821 resulted in a dose-dependent reduction in viability, whereas VE-821 has the most efficient response. Quantification of γH2AX phosphorylation and protein expression of the DNA repair pathways showed a reduced regenerative capacity after irradiation. Furthermore, combined treatment with VE-821 and particle irradiation increased MAPK phosphorylation and the expression of apoptosis markers. At the gene expression and at the protein expression/phosphorylation level, we were able to demonstrate the preservation of DNA damage after combined treatment. The present data showed that the combined treatment with ATMi VE-821 increases the radiosensitivity of human chondrosarcoma cells in vitro and significantly suppresses efficient DNA repair mechanisms, thus improving the efficiency of radiotherapy.

Cellular and Molecular Biological Alterations after Photon, Proton, and Carbon Ions Irradiation in Human Chondrosarcoma Cells Linked with High-Quality Physics Data.

Chondrosarcomas are particularly difficult to treat due to their resistance to chemotherapy and radiotherapy. However, particle therapy can enhance local control and patient survival rates. To improve our understanding of the basic cellular radiation response, as a function of dose and linear energy transfer (LET), we developed a novel water phantom-based setup for cell culture experiments and characterized it dosimetrically. In a direct comparison, human chondrosarcoma cell lines were analyzed with regard to their viability, cell proliferation, cell cycle, and DNA repair behavior after irradiation with X-ray, proton, and carbon ions. Our results clearly showed that cell viability and proliferation were inhibited according to the increasing ionization density, i.e., LET, of the irradiation modes. Furthermore, a prominent G2/M arrest was shown. Gene expression profiling proved the upregulation of the senescence genes CDKN1A (p21), CDKN2A (p16NK4a), BMI1, and FOXO4 after particle irradiation. Both proton or C-ion irradiation caused a positive regulation of the repair genes ATM, NBN, ATXR, and XPC, and a highly significant increase in XRCC1/2/3, ERCC1, XPC, and PCNA expression, with C-ions appearing to activate DNA repair mechanisms more effectively. The link between the physical data and the cellular responses is an important contribution to the improvement of the treatment system.

Cytosolic nucleic acid sensors and interferon beta-1 activation drive radiation-induced anti-tumour immune effects in human pancreatic cancer cells.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related deaths worldwide with limited treatment options due to extensive radiation and chemotherapy resistance. Monotherapy with immune checkpoint blockade showed no survival benefit. A combination of immunomodulation and radiotherapy may offer new treatment strategies, as demonstrated for non-small cell lung cancer. Radiation-induced anti-tumour immunity is mediated through cytosolic nucleic acid sensing pathways that drive the expression of interferon beta-1 (IFNB1) and proinflammatory cytokines. Methods: Human PDAC cell lines (PANC-1, MIA PaCa-2, BxPC-3) were treated with X-rays and protons. Immunogenic cell death was measured based on HMGB1 release. Cytosolic dsDNA and dsRNA were analysed by immunofluorescence microscopy. Cell cycle progression, MHC-I and PD-L1 expression were determined by flow cytometry. Galectin-1 and IFNB1 were measured by ELISA. The expression levels and the phosphorylation status of the cGAS/STING and RIG-I/MAVS signalling pathways were analysed by western blotting, the expression of IFNB1 and proinflammatory cytokines was determined by RT-qPCR and genome-wide by RNA-seq. CRISPR-Cas9 knock-outs and inhibitors were used to elucidate the relevance of STING, MAVS and NF-κB for radiation-induced IFNB1 activation. Results: We demonstrate that a clinically relevant X-ray hypofractionation regimen (3x8 Gy) induces immunogenic cell death and activates IFNB1 and proinflammatory cytokines. Fractionated radiation induces G2/M arrest and accumulation of cytosolic DNA in PDAC cells, which partly originates from mitochondria. RNA-seq analysis shows a global upregulation of type I interferon response and NF-κB signalling in PDAC cells following 3x8 Gy. Radiation-induced immunogenic response is regulated by STING, MAVS and NF-κB. In addition to immunostimulation, radiation also induces immunosuppressive galectin-1. No significant changes in MHC-I or PD-L1 expression were observed. Moreover, PDAC cell lines show similar radiation-induced immune effects when exposed to single-dose protons or photons. Conclusion: Our findings provide a rationale for combinatorial radiation-immunomodulatory treatment approaches in PDAC using conventional photon-based or proton beam radiotherapy.